We have demonstrated that the chemoprotective effect of fish oil (induction of apoptosis, suppression of both cell proliferation and colon tumor formation) is enhanced when a highly fermentable fiber (pectin), capable of elevating lumenal butyrate levels, is added to the diet. From a mechanistic standpoint, butyrate is a Histone Deacetylase inhibitor (HDACi) known to alter chromatin structure. This is noteworthy, because global changes in chromatin structure, i.e., epigenetic landscape, are a hallmark of cancer. Since transformation of adult stem cells is an extremely efficient route towards initiating intestinal cancer, an overall goal of this proposal is to use novel Lgr5-LacZ and Lgr5-EGFP-IRES-cre knockin mouse models to examine the potential of combined dietary fish oil and pectin treatment to modulate adult intestinal stem cell- specific targeted apoptosis, changes in chromatin structure and non-coding RNA/gene expression profiles. This goal is supported by our recent observation that combined fish oil and pectin treatment modulates a subset of non-coding microRNAs and their target genes (mRNAs) implicated in the regulation of the colon stem cell niche and tumor evolution. Aim 1 will quantify the number and spatio-temporal location of stem cells, DNA damage and targeted apoptosis in the colonic crypt at the initiation and tumor stages of colon carcinogenesis following exposure to chemoprotective diets containing fish oil and pectin. We have hypothesized that dietary fish oil and pectin combination modulates stem cell DNA damage by enhancing targeted apoptosis, in part, by suppressing regulators of stem cell self-renewal. Aim 2 will compare high- resolution chromatin-state maps for the promoters of genes that respond to fish oil/pectin/carcinogen co- treatment in mouse colonic stem cells. We have hypothesized that fish oil modulates butyrate-induced chromatin dynamics/plasticity in the colon. Aim 3 will further characterize stem cell non-coding microRNAs (and their targets), known to be modulated by fish oil/pectin/carcinogen co-treatment, in HCT116 human colon cancer cells and a mouse organoid stem cell model. We have hypothesized that fish oil and pectin combination uniquely suppresses growth of colonocytes/crypts by antagonizing the expression of genes which regulate stem cell signaling. The proposed experiments are highly novel and relevant because the impact of colorectal cancer chemoprevention agents on adult intestinal stem cell biology has not been determined. PUBLIC HEALTH RELEVANCE: We have demonstrated that the bioactive components generated by fish oil (n-3 PUFA) and fermentable fiber (butyrate) act coordinately to protect against colon cancer. The proposed studies will determine the epigenetic therapeutic potential of combined fish oil and pectin treatment on intestinal stem cel targeted deletion and chromatin plasticity/dynamics.